A class of non-histone proteins from HeLa cells which stays stably associated with chromatin DNA in 2.5M NaCl-5M urea solution has been described previously (Pederson and Bhorjee, 1975). It is proposed to identify and investigate the nature of the DNA-binding sites for the tight proteins, and the stoichiometry of binding using cross-linking agents. Specifically, HeLa DNA-tight protein complex will be isolated and cross-linked by exposure to ultraviolet light. The UV-cross-linked DNA sequences will be retrieved by alternate nuclease and proteinase treatment, nick-translated and their repetitive and non-repetitive sequence composition analyzed from the reassociation kinetics data. Using the reversible protein cross-linker dithiobis (succinimidyl propionate), the homotypic or heterotypic interaction of the tight proteins in chromatin will be analyzed by SDS-polyacrylamide gel electrophoresis of the cross-linked products. The evolutionary conservation of the tight protein non-histones obtained from divergent organisms: HeLa, Drosophila cultured cells, and Dictyostelium, will be examined by comparing them electrophoretically and immunochemically. To visualize the cytological distribution of the tight protein non-histones, chromosomal localization of the Drosophila tight proteins will be examined by in situ immunofluorescence in polytene chromosomes.